The linear relationship between the change in the oscillation frequency of a piezoelectric crystal and the mass variation on the crystal as a result of binding or absorption phenomena, has been used to advantage in a gravimetric monitoring of antigen-antibody binding. The mathematical relationship between the frequency changes of a piezoelectric crystal, Δf, and mass changes, Δm, on the crystal is given by the following Sauerbrey equation:Δf=−2.3×106fo2·Δm/Awhere fo is the fundamental resonance frequency of the crystal prior to the mass variation and A is the surface area of deposited mass. For example, for a crystal exhibiting a fundamental frequency of 9 MHz and surface area of 1 cm2, a mass-change on the crystal that corresponds to 1×10−9 g, will stimulate a frequency change, Δf, of 6 Hz.
The first analytical use of piezoelectric crystals in relation to antigen-antibody (Ag-Ab) interactions was reported in 1972(1), where a nyebar precoated crystal was further coated via hydrophobic interactions, with bovine serum albumin (BSA) and the association of the BSA-Ab to the crystal was monitored by the frequency changes. Since then, the piezoelectric detection of antigens and antibodies by piezoelectric means or the quartz crystal microbalance (QCM) has been adopted in a series of analytical studies. The progress in this area has been reviewed by Suleiman et al., 1994(2) and Ward et al., 1990(3).
Several patents describe the application of QCM for the analysis of antigens and antibodies. Physical adsorption of antigens to a crystal was used as a means for the detection of antigens by interacting the crystal with a mixture of the analyte antigen and a predetermined amount of Ab(4). The decrease in the antigen concentration was inversely related to the antigen concentration in the sample. In two patents by Rice(5.6), methods for the determination of Abs by QCM were disclosed. The antigen was immobilized on a polymer precoated crystal and the frequency changes as a result of Ab association related to the analyte Ab concentration in the sample. By this method, human IgG against honey bee venom, phospholipase A, and keyhold limpet hemocyanine were analyzed(7). However, non-specific binding to the crystal interfered with the analyses. In a follow-up patent(8), the detection of low molecular weight components by a pre-coated crystal with the anti-Ab and competitive binding assay of the Ab-low molecular weight analyte was described. All of these analyses were performed by treatment of the crystals in solution and subsequent frequency measurements in air. This two-step solution/gas procedure allows improvement of the sensitivity of the resonating QCM, but introduces technical complications and the interference of hydration/dehydration phenomena that are reflected in the frequency parameters.
Piezoelectric immunoassaying in the liquid phase has important technical advantages as it allows stationary and flow analysis of aqueous samples. The method suffers, however, from a basic physical limitation due to substantially lower frequency changes of the crystal as a result of the solution viscosity. QCM immunoassays in solution were reported by Roederer(7) and addressed in a follow-up patent(8). The quartz crystal was modified with glycidoxypropyltrimethoxy silane (GOPS), and the surface-modified crystal was then further modified by anti-human IgG antibody and then applied for the piezoelectric detection of human IgG. The detection limit of the device was determined to be 13 μg·ml−1. A closely related approach was adopted by Muramatsu et al.(9) where the quartz crystals were surface-modified by γ-aminopropyl triethoxy silane and further derivatized by protein A. The surface-modified crystals were then applied for the determination of human IgG in the concentration range 10−6–10−2 mg·ml−1. A related patent disclosed the piezoelectric analysis of thyroxine using a polyamide 6 polymer coating and anti-thyroxine Ab as sensing interface(10).
Piezoelectric analysis of high molecular weight antigens such as microbial cells was addressed using antibody-coated quartz-crystals. C. albicans cells in the concentration range 1×106–5×108 cells·ml−1 were analyzed by an anti-Candida albicans Ab surface(11), E. coli with an anti-E. coli interface(12) and protein A-coated crystals acted as piezoelectric sensing interface for various bacteria including Salmonella, Shigella, Yersinia and E. coli(13). Various schemes allowing efficient use of QCM in assaying analytes in a liquid media have been described in WO 97/04314(14).
QCM techniques were used hitherto only for detection of interaction with substances of relatively high molecular weight, e.g. proteins and other macromolecules. The QCM techniques are generally considered not sufficiently sensitive for detection of substances of a low molecular weight.